Purpose: :
Efficient in vitro production is a critical hurdle in the large-scale manufacture of PPCs for future pre-clinical and clinical stem cell trials in retinal degeneration. We proposed to enhance published protocols by reducing the time taken for differentiation whilst also increasing yield of PPCs for a given number of hESCs. Differentiation protocols have previously focused on inhibiting pro-growth pathways (e.g., Wnt, BMP, and Nodal), and small-molecule targeted induction agents (e.g., thyroid hormone, retinoic acid, FGF, and IGF). We hypothesize that driving expression of CRX (a key transcription factor in photoreceptor differentiation) during directed hESC differentiation will enhance production of PPCs. We propose to achieve this through synthetic-mRNA induction.

Methods: :
We adapted established PPC-production methods by, firstly, using size-controlled embryoid bodies (EBs) to synchronize hESC differentiation and, secondly, by using TeSR2 xeno-free media in our feeder-free differentiation protocol. We have developed a CRX-encoding expression vector for in vitro transcription, with CRX transcripts ‘capped’ with 7-methylguanosine and treated with phosphatases. Lipoplexes were used to transfect cells with this CRX mRNA. Following this, immunocytochemistry and RT-PCR was used to assess gene expression in differentiating hESCs.

Results: :
Size-controlled EBs significantly improved PPC yield, as 10,000-cell EBs exhibited a more complete reduction of residual-hESC colony formation relative to 1,000-cell and random-size EBs. We have also calculated the benefits of introducing CRX-mRNA into differentiation media at a stage when cells express PAX6 and MITF through NRL and Chx10 expression analysis.